Which microscope can image GFP and RFP in the same live-cell experiment in a UK lab?
One of the most common fluorescence-microscopy questions in research forums is how to image GFP and RFP — or GFP and mCherry — in the same live cells. The question usually sounds like: “I need to see two fluorescent reporters in my cell culture at the same time. What microscope do I actually need?”
This guide answers that for UK labs. It covers the hardware that matters, the mistakes that cause bleed-through, and the EVOS systems that handle GFP/RFP imaging at benchtop scale.
At minimum, a dual-colour live-cell fluorescence setup needs:
You do not need confocal for every GFP/RFP experiment. Widefield fluorescence with the right filters and camera is enough for transfection checks, reporter assays, co-cultures and many co-localisation studies.
GFP excites around 488 nm and emits around 509 nm. RFP variants vary, but mCherry — the most common red partner — excites around 587 nm and emits around 610 nm. That 100 nm gap is large enough that a standard GFP/RFP filter pair gives clean separation if the filters are narrow.
Older “DsRed” or generic RFP variants have broader spectra that can tail into the GFP channel. If your red construct is unspecified, switching to mCherry (or mScarlet/mRuby) usually makes dual imaging easier.
Rule of thumb: if you can choose the red fluorophore, use mCherry with GFP. The pair is well separated, widely available, and easy to filter.
| Platform | Best For | Fluorescence Channels | Live Cell? |
|---|---|---|---|
| EVOS M3000 | Entry-level GFP/RFP checks, basic transfection imaging | Single + dual fluorescence capable | Yes, benchtop |
| EVOS M5000 | Four-colour routine fluorescence, larger field of view, stitched tiles | Up to 4 fluorescence channels (GFP, RFP, DAPI, CY5) | Yes, benchtop |
| EVOS M7000 | Automated time-lapse, Z-stacks, multi-well plates, long experiments | Multi-channel fluorescence + transmitted light | Yes, with on-stage incubation |
The EVOS M3000 is a compact, all-in-one inverted fluorescence microscope. It is the simplest way for a UK lab to start imaging GFP and RFP without building a traditional epifluorescence rig around a research microscope body.
It suits routine checks: “Did my cells take up the GFP construct? Is the RFP reporter expressed? Are the two populations mixed?” It is not designed for overnight time-lapse or automated multi-well screening.
If your GFP/RFP work expands to include DAPI, CY5, or if you want to capture larger stitched fields, the EVOS M5000 is the logical upgrade from the M3000.
It remains a benchtop system, so it is accessible to individual groups without core-facility booking queues.
When the experiment becomes “watch GFP and RFP over hours or days and measure how the ratio changes,” the EVOS M7000 is the relevant EVOS platform.
This makes it useful for FRET reporters, proliferation assays, cell competition experiments and any assay where the GFP/RFP ratio changes over time.
Bleed-through is the most common complaint when people start dual-colour imaging. It happens when signal from one fluorophore appears in the other channel. Typical causes and fixes:
| Cause | Fix |
|---|---|
| Broad RFP variant tails into GFP channel | Use mCherry or mScarlet instead of older DsRed-type proteins |
| Filters are too wide | Switch to narrower bandpass emission filters |
| One channel is massively overexposed | Reduce exposure/gain for the bright channel first |
| Simultaneous excitation excites both fluorophores | Use sequential capture with single-band cubes |
| Autofluorescence looks like signal | Include unstained and single-colour controls |
Hickey & Ung, Cells 2021 — practical guide to microscope hardware, fluorophore choice and live-cell imaging parameters.
DOI: 10.3390/cells11010035Shaner et al., Nature Biotechnology 2004 — the mCherry/mFruit series paper that made red fluorescent proteins reliable for dual-colour work.
DOI: 10.1038/nbt1174Phillips et al., Wellcome Open Research 2018 — free web tool to check filter and fluorophore compatibility before imaging.
DOI: 10.12688/wellcomeopenres.14703.1Product page for the entry-level benchtop fluorescence microscope for GFP/RFP and single-colour work.
Thermo Fisher UKFour-colour benchtop fluorescence microscope for routine multi-channel imaging in UK labs.
Thermo Fisher UKAutomated inverted fluorescence microscope for live-cell time-lapse, Z-stacks and multi-well GFP/RFP assays.
Thermo Fisher UKYou need an inverted fluorescence microscope with separate GFP and RFP filter cubes or a multi-band filter set, a sensitive sCMOS/CMOS camera, and enough stage clearance for your culture vessel. For routine benchtop work the EVOS M3000 or M5000 capture GFP/RFP pairs without a darkroom. For automated time-lapse, Z-stacks or multi-well plates the EVOS M7000 is the next step up.
Yes, if your microscope has a dual- or multi-band excitation/emission filter set that covers both fluorophore bands in one shot. Sequential capture with single-band GFP and RFP cubes is more common because it gives the cleanest separation and avoids bleed-through, especially when one fluorophore is much brighter than the other.
Not always. Widefield fluorescence with properly matched filters and a sensitive camera is enough for many co-localisation, transfection-efficiency and reporter assays. Confocal helps when samples are thick, when out-of-focus blur is hiding detail, or when you need quantitative optical sectioning.
EVOS M3000 is the entry-level benchtop fluorescence system for single-colour and basic dual-colour work. EVOS M5000 adds four-colour fluorescence, a larger monitor and more flexible stage. EVOS M7000 adds automation, time-lapse, Z-stacks, on-stage incubation and multi-well scanning for longer or more complex GFP/RFP experiments.
Bleed-through happens when the excitation or emission spectra of GFP and RFP overlap, or when a fluorophore is so bright that its tail spills into the neighbouring detector band. Fixes include narrower filters, sequential capture, shorter exposure for the bright channel, spectral unmixing, and choosing fluorophores with better-separated spectra such as mCherry instead of older RFP variants.
GFP with mCherry is one of the most popular pairs because their excitation/emission peaks are well separated (GFP ~488/509 nm, mCherry ~587/610 nm). This reduces bleed-through and simplifies filter selection compared to older RFP variants that have broader spectra closer to GFP.